Membrane hyperpolarization activates trout sperm without an increase in intracellular pH

Sperm cells are terminally differentiated cells with a limited number of cellular functions, which include initiation and regulation of motility. Most sperm cells are immotile in the seminal tract and become motile in a fertilization environment. In this study we investigated the mechanism by which trout sperm recognize the signal to activate and initiate motility. Trout sperm, like most teleost sperm, lack an acrosome, thereby eliminating interference from acrosomal functions in the assessment of motility-specific functions. Immediate and synchronous activation of trout sperm motility upon dilution into a fertilizing environment allows for quantitative assessment of activation-related functions correlated with motility. Thus, trout sperm is a good model for studying initiation and regulation of sperm motility.

Although it has been known since 1938 that K⁺ is primarily responsible for keeping trout sperm inactive in the seminal tract (Schlenk and Kahmann, 1938), it was not clear how K⁺ regulated initiation of movement. In most animal cells, including sea urchin sperm, the resting membrane potential is primarily set by K⁺ permeability (Schackmann et al. 1984). Recently it has been shown that K⁺ can contribute to the resting membrane potential in trout sperm under long-term incubation (Gatti et al. 1990).

Thus, it is possible that K⁺ regulation is mediated through changes in the membrane potential. Influx of Ca²⁺ (Tanimoto and Morisawa, 1988; Cosson et al. 1989), increases in intracellular [cyclic AMP] (Benau and Temer, 1980; Morisawa and Ishida, 1987), and changes in intracellular pH (Robitaille et al. 1987) have also been reported to contribute to the activation of trout sperm. In addition, an increase in intracellular pH has been suggested to be a conserved mechanism for activation of sperm motility (Babcock and Pfeiffer, 1987; Shapiro and Tombes, 1989). In this study, we investigated the initial steps in the activation of trout sperm motility, particularly the roles of membrane potential and intracellular pH.

All solutions were made from reagent grade chemicals obtained from Mallinkrodt, Sigma and Baker Chemical Companies. Carboxyfluorescein diacetate (CFDA), and 3,3’-dipropylthiocar-bocyanine iodide (diS-C3-(5)) were obtained from Molecular Probes, Eugene, OR. Valinomycin, nigericin, digitonin and l-anilino-8-naphthosulfonate (ANS) were obtained from Sigma Chemical Company.

All activation solutions (AS: 150 nun NaCl, 20 nun glycine, 10 mH Tris–HCl, pH 8.7) activate full movement of trout sperm, while all non-activation solutions (NAS) do not activate trout sperm motility. The compositions of NAS differed slightly for different experimental purposes and are listed below.

In the divalent activation experiments, all motility test solutions had 20 mw glycine, 10 mw Tris–HCl, pH 8.7, variable concentrations of KC1, and a total osmolality adjusted to 330mosmol with NaCl. Solutions with divalent cations had, in addition, 2 mM EGTA. Calcium and magnesium were added from stock solutions as their chloride salts while cadmium was added as the acetate. [Free Ca²⁺] and [free Mg²⁺] were calculated using the algorithm described by Brokaw (1986) and association constants reported by Sillen and Martell (1964). [Free Cd²⁺] was estimated by subtracting [total EGTA] from [total Cd²⁺].

Solutions used to test the effect of external pH contained 2mM EGTA and an appropriate buffer for each pH tested: piperazine-N, N′-bis(2-ethanesulfonic acid) (Pipes), pH6.5–7.0; N-2-hydroxethylpiperazine-N′-2-ethanesulfonic acid (Hepes), pH 7.0–8.0; tris(hydroxymethyl)amino-methane (Tris) pH 7.5–9.0; or 3-(N-Tris-(hydroxymethyl)methylamino)-propane sulfonic acid (Taps) for pH8.5–9.5. Osmolality was kept constant in all solutions by the addition of NaCl (total osmolality= 332mosM). AS contained no K⁺, NAS and divalent cation solutions contained 30 mM K⁺. Mg²⁺ solution contained, in addition, 18 mM free Mg²⁺, and Ca²⁺ solution contained 8 TUM free Ca²⁺.

Seminal mimic solution (SM) contained the following: 127 mM NaCl, 1.5mM MgCl₂, 37 mM KCH₃CO₂, 30mM Hepes, and pH as stated in text. Cesium seminal mimic (CsSM) contained the following: 97 mM NaCl, 27 mM NalCH₃CO₂), 10 mM K(CH₃CO₂), 37mM CsCl, 1.5mM MgCl₂, 2.6mM CaCl₂, 30mM Hepes, pH7.2.

Extender solution contained the following: 32 mM KC1, 108 mM NaCl, 12 mM NaHCO₃, 4mM NaH₂PO₄, 1.0 mM MgSO₄, 1.0 mM CaCl₂, 0.1% glucose, 1160 i.u.ml⁻¹ streptomycin and 1250i.u.ml⁻¹ penicillin.

diS-C₃-(5) was stored in absolute ethanol at a concentration of 0.2 M.

ANS was stored in NAS at a concentration of 2 M.

Sperm were obtained from Oncorhynchus mykiss (Rainbow and Steelhead trout) maintained at the Washington State University Campus Fish Center. The trout were anesthetized with 2-phenoxyethanol and gently squeezed along the abdomen to express sperm. Sperm were collected in a sealable plastic bag and immediately stored between 4 °C and 10 °C. The sperm were later oxygenated and/or put into Extender solution for further storage at 4°C. Other steelhead sperm were obtained from the Dworshak National Fish Hatchery in Ahsahka, Idaho, by similar techniques.

For determination of percentage motility, 1 μl sperm was diluted into 1 ml of the appropriate test solution and observed under darkfield microscopy on a temperature controlled stage at 10°C. Motility estimates were repeated a minimum of twice on each sample (minimum of 200 sperm/sample). Sperm motility estimates were later confirmed using video microscopy and Expert Vision Motion Analysis Systems (Santa Rosa, CA).

A 100 pl sample of sperm was incubated with 200 μl of appropriate SM, pH 7.2, in an Eppendorf tube for 15 min on ice with gentle agitation. The mixture was centrifuged for 4 s (∼10000 g), the supernatant was discarded, and the pellet resuspended in SM, pH 7.2, unless otherwise stated in the text. Centrifugation and resuspension were repeated once. The sperm solution was then allowed to equilibrate in SM for 10 min at 10 °C.

diS-C₃-(5) in absolute ethanol at a concentration of 0.2 M was added to sperm in 2 ml of NAS (85 mM NaCl, 15 mM KC1, 20 mM glycine, 10 mM Tris–HCl, pH 8.7, at 4×10⁶ to 6×10⁶ cells ml⁻¹) to a final concentration of 0.25 pM diS-C₃-(5). K⁺, Cs⁺ and Mg²⁺ were added from a 1 M stock of their chloride salts to the final concentrations listed in Fig. 1. diS-C₃-(5) fluorescence was monitored at 620 nm and A,_m 670 nm with continuous stirring with a glass-coated stir bar at 10°C. diS-C₃-(5) loading alone did not significantly change the motility of the trout sperm.

ANS in NAS at a concentration of 2 M was added to sperm in 2 ml of NAS (30 mM KC1, 70 mM NaCl, 20 mM glycine, 10 mM Tris–HCl, pH 8.7, at 4×10⁶ to 6×10⁶ cells ml⁻¹) to a final concentration of 15 pM ANS. K⁺, Cs⁺ and Mg²⁺ were added from 1M stocks of their chloride salts to the final concentrations listed in Fig. 4. ANS fluorescence was monitored at λ_ex 366 nm and λ_em 475 nm with continuous stirring at 10°C. ANS at the concentration used had no effect on sperm motility. The addition of K⁺, Cs⁺ or Mg²⁺ without cells had no effect on ANS fluorescence.

Carboxyfluorescein (CF) was generated intracellularly by the incubation of approximately 100 pl of trout sperm with 200 μl of 2 μM carboxyfluorescein diacetate (CFDA) in SM, pH 6.5 (to result in a final sperm concn between 4×10⁶ and 6×10⁶ cells ml⁻¹), in an Eppendorf tube for 15 min on ice with gentle agitation. This mixture was centrifuged for 4 s (∼10 000 g), the supernatant was discarded, and the pellet resuspended in SM, pH 7.2, unless otherwise stated in the text. Centrifugation and resuspension were repeated once to eliminate extracellular carboxyfluorescein. The sperm solution was then allowed to equilibrate in SM for 10 min at 10°C.

A 10 μl sample of the CF-loaded sperm solution was added to 2 ml of NAS and CF fluorescence was monitored at λ_ex 492 nm and λ_em 516 nm, with continuous stirring at 10°C. Fluorescence measurements were normalized to the maximal fluorescence obtained by release of CF by digitonin (final concn 50 μg ml⁻¹) and raising the pH of the solution to approximately pH 10 with 25 μl 2 M NaOH.

For the manipulation of intracellular pH, NAS consisted of 20 mM KC1, 80 mM NaCl, 20 mM glycine, and 10 mM Tris–HCl, pH 8.7; except for nigericin, which contained 150 mM KC1, 20 mM glycine, and 10 mM Tris–HCl, pH 8.7. Upon equilibration of the fluorescence signal (approximately 1 min), acid or base was added to a final concentration of 10 mM. For the measurement of intracellular pH during divalent cation activation NAS consisted of 20 mM KC1, 80 mM NaCl, 20 mM glycine, and 10 mM Tris–HCl, pH 8.7. Sperm were activated after equilibration of the fluorescence signal with the addition of Mg²⁺ to a final concn of 10 mM. F_max was obtained at pH 8.7, due to the lack of solubility of Mg²⁺ at pH 10.0. The absolute value of the pH change was interpolated from null point measurements using ΔF/F_max.

Trout sperm can be activated by lowering the external [K⁺]. Once activated, the subsequent increase in external [K⁺] to inhibitory levels within 1 s did not inhibit motility. In order to determine if K⁺ contributes to membrane potential in trout sperm on a time scale necessary for this rapid and irreversible activation, we directly monitored membrane potential with the fluorescent probe 3,3′-dipropylthiocarbocyanine iodide (diS-C₃-(5)) (Fig. 1). The positively charged diS-C₃-(5) is redistributed across the lipid bilayer of the membrane, driven by the membrane potential (Cabrini and Verkman, 1986). Hyperpolarization of the membrane decreases diS-C3-(5) fluorescence, while depolarization increases its fluorescence. Upon addition of external K⁺, the fluorescence of diS-C3-(5) immediately increased, indicating a depolarization of the membrane. Valinomycin, an ionophore that transports K⁺ and Cs⁺ across biological membranes (Pressman et al. 1967), does not alter diS-Cs-CS) fluorescence to increased external [K⁺] (Fig. 1A). Cs⁺ is not membrane permeant and does not contribute to the membrane potential as indicated by the lack of change in the diS-C3-(5) fluorescence upon the addition of Cs⁺ to the external medium (Fig. IB). However, with the addition of valinomycin, Cs⁺ becomes membrane permeant. In the presence of valinomycin the fluorescence of diS-C₃-(5) increased upon the addition of Cs⁺ (Fig. IB), indicating that in the presence of valinomycin, Cs⁺ contributes to the membrane potential.

To investigate whether changes in membrane potential regulate trout sperm motility, we observed the behavior of sperm whose membrane potential was manipulated by the ionophore valinomycin and Cs⁺ (Fig. 2). Cs⁺ at the concentration used does not reduce the percentage motility of untreated sperm. The motility of sperm suspended in a solution that mimics the seminal plasma (SM) still retains significant motility in the presence of Cs⁺. The absence of inhibition of motility by Cs⁺ is expected, since Cs⁺ alone does not contribute to the membrane potential. In the presence of valinomycin (SM+Val; CsSM+Val), Cs⁺ dramatically reduces the percentage activation under conditions that normally fully activate trout sperm (0K⁺, 30Cs⁺). This result is consistent with an inward Cs⁺ current, depolarizing the membrane and inhibiting activation. Sperm preincubated in CsSM with valinomycin are activated upon the dilution of external Cs⁺, even in the presence of a normally inhibitory [K⁺] (15K⁺, 0Cs⁺). Thus, an outward Cs⁺ current can overcome the inhibitory K⁺ gradient, hyperpolarize the membrane, and activate motility. These effects of Cs⁺ in the presence of valinomycin are consistent with a depolarization inhibiting and a hyperpolarization triggering activation.

In addition to activation by the dilution of external [K⁺], trout sperm can be activated in the presence of normally inhibitory [K⁺] by the addition of the divalent cations. In Fig. 3 we show activation of trout sperm in normally inhibitory [K⁺] by the addition of the divalent cations Ca²⁺, Mg²⁺ and Cd²⁺. In addition, Mn²⁺ can also overcome K⁺ inhibition in a range of concentration similar to that of Mg²⁺ (data not shown). The concentration of divalent cations needed to activate motility is dependent upon the external [K⁺], The divalent cations’ ability to overcome K⁺ inhibition can be interpreted as a masking of membrane surface potential to provide a hyperpolarizing environment to membrane voltage sensors (see Fig. 7, below). We can estimate the surfacecharge masking effect of divalent cations from the plot of the concentration of divalent cations on a log scale required to overcome K⁺ inhibition, plotted as the Nemst Potential for K⁺ (Fig. 3). The interpretation of the effects of divalent cations on the membrane in terms of surface charge screening predicts a potential change of 27 mV per 10-fold change in divalent cation concentration (McLaughlin et al. 1971). The plot in Fig. 3 shows, as predicted, a roughly linear relationship between the calculated K⁺ equilibrium potential versus log of divalent cation concentration, with somewhat higher slopes (33 mV per 10-fold concentration change for Mg²⁺ and 42 mV per 10-fold concentration change for Ca²⁺). These higher values may be due to an error in the estimate of intracellular [K⁺] or to a contribution to the membrane potential from other ions, principally Cl⁻. Theoretically, trivalent cations should be 10 times more effective than divalent cations in masking surface charge (McLaughlin et al. 1971). We found that sub-mw concentrations of La³⁺ activate trout sperm in the presence of inhibitory [K⁺]. However, La³⁺ also causes massive clumping of the cells, and prevents quantification of activation.

We directly monitored the surface potential of trout sperm upon addition of divalent and monovalent cations with the fluorescent surface binding probe, l-anilino-8-naphthosulfonate (ANS). The fluorescence of ANS is enhanced in a hydrophobic environment such as the membrane (Vanderkooi and McLaughlin, 1976). The negatively charged headgroups on the membrane phospholipids reduce the binding of ANS and the subsequent fluorescence enhancement. Upon the addition of an agent that screens surface charges of the membrane, Mg²⁺, we observed an increase in the fluorescence of ANS (Vanderkooi and McLaughlin, 1976) (Fig. 4A),which is dependent on [Mg²⁺] (Fig. 4B) as expected (Fig. 3). In contrast, the addition of the monovalent ions, Cs⁺ or K⁺, actually decreased ANS fluorescence due to dilution. These results are consistent with divalent cations having an approximately 10-fold higher capacity to mask the surface charge than monovalent cations (Vanderkooi and McLaughlin, 1976; McLaughlin et al. 1971).

In Fig. 5, we show that percentage motility of trout sperm in divalent cation-supplemented K⁺ solutions, as well as in K⁺-free solution, is pH dependent. The fact that trout sperm activation is inhibited at low external pH is consistent with the hypothesis that an increase in intracellular pH is necessary for activation. Thus, we tested to see if increasing intracellular pH can activate trout sperm motility. We increased the intracellular pH of trout sperm by two methods. The addition of 10 mM NH4CI increased the intracellular pH, but did not activate trout sperm in the presence of an inhibitory [K⁴] (Fig. 6A). An alternative method of raising the intracellular pH, using the K⁺/H⁺ exchanger, nigericin, in the presence of high external [K⁺] at pH 8.7 (Babcock, 1983), also increased intracellular pH, but did not activate motility (Fig. 6B). Therefore, raising intracellular pH is not sufficient to activate trout sperm. Furthermore, upon activation there is an acidification of approximately 0.19 pH unit, rather than an alkalinization (Fig. 6C). A similar magnitude of acidification induced by weak acids did not activate trout sperm in the presence of inhibitory [K⁺] (Fig. 6D). The time course of acidification suggests that it is a result of, rather than the trigger for, activation. All of the above manipulations of intracellular pH were not deleterious to the trout sperm, since the treated sperm were activated upon subsequent dilution into an activation solution.

Our work shows the importance of membrane potential to the regulation of the activation of trout sperm. Our direct measurement of membrane potential demonstrates that K⁺ contributes to rapid changes in the membrane potential of trout sperm. By manipulating the membrane potential of trout sperm with Cs⁺ and the ionophore valinomycin we regulate sperm activation independently of [K⁺]. Thus, K⁺ regulation of trout sperm activation is mediated through its effect on the membrane potential.

Gatti and coworkers (1990) used slow lipophilic tracers to show that K⁺ contributes to changes in the membrane potential. However, measurement of membrane potential using lipophilic tracers is on a time scale of minutes. Since trout sperm are irreversibly activated in less than 1 s and cease to move within 1–2 min, the relevance of changes at these time scales to activation is unclear. Tanimoto and Morisawa (1988) used K⁺-channel blockers to inhibit activation in a K⁺-free activation solution. They concluded from this result that activation is dependent on a K⁺ efflux, and thus membrane potential changes. Our measurements show that K⁺ contributes to bulk potential changes on a time scale relevant to activation and that membrane potential changes can regulate activation independently of K⁺.

Divalent cation addition can also overcome K⁺ inhibition of trout sperm. Although previous authors have mentioned the antagonistic effects of Ca²⁺ on K⁺ inhibition of activation (Schlenk and Kahmann, 1938; Baynes et al. 1981; Tanimoto and Morisawa, 1988; Cosson et al. 1989), no unifying mechanism has been proposed to explain other divalent cations’ abilities to overcome K⁺ inhibition. Tanimoto and Morisawa (1988), and Cosson and coworkers (1989) have proposed that calcium activation in the presence of inhibitory [K⁺] involves calcium influx. However, such a mechanism does not explain the ability of other divalent cations to overcome K⁺ inhibition. We show that divalent cations can mask the surface potential of trout sperm membranes. We propose a model to explain the contribution of the surface potential to activation of sperm motility (Fig. 7; Hille et al. 1975). In this model, masking of the surface charge would cause a hyperpolarization to be sensed by the membrane voltage sensor. Both bulk potential changes and surface potential changes can contribute to a sensed hyperpolarization of a membrane voltage sensor and trigger activation.

We directly measured the intracellular pH of trout sperm at rest and upon activation. Trout sperm undergo an acidification upon activation. A similar magnitude of acidification induced by weak acids does not activate trout sperm in the presence of inhibitory [K⁺]. Robitaille and coworkers have previously measured changes in intracellular pH of trout sperm after activation using ³¹P NMR (Robitaille et al. 1987). The time scale of measurements using ³¹P NMR does not allow for measurement of changes in pH that are physiologically relevant to trout sperm activation. In other sperm, including those of sea urchin (Lee et al. 1983) and mammals (Wong et al. 1981; Babcock et al. 1983), alkalinization of intracellular pH induces activation. This alkalinization has been suggested to be a conserved mechanism of activation of sperm motility (Babcock and Pfeiffer, 1987; Shapiro and Tombes, 1989). However, trout sperm activation is clearly not regulated by changes in intracellular pH. Thus, intracellular pH change cannot be a conserved step in the activation of sperm motility.

We have shown that trout sperm can be activated by three different methods (by dilution of external K⁺, addition of divalent cations or hyperpolarization with Cs⁺ and valinomycin) that affect the sensed potential in the membrane and we suggest that hyperpolarization sensed by membrane voltage sensors is the initial trigger for activation. This hyperpolarization leads to activation of motility without mediation by intracellular pH. It is interesting to note that different mechanisms of alkalinization of intracellular pH that activate sea urchin, ram and bull sperm are regulated by membrane potential (Babcock and Pfeiffer, 1987; Babcock et al. 1983; Lee, 1984). How membrane potential changes affect the intracellular environment in order to activate trout motility remains to be elucidated.

A Shimadzu spectrofluorimeter RF-540 was generously provided by Dr L. Randall of Biophysics/Biochemistry Program, WSU. We are grateful to Dr Gary Thorgaard, Paul Wheeler and Paul Scheerer for providing us with trout sperm. Sperm samples were also generously provided by Dworshak National Fish Hatchery and South Tacoma Hatchery. This investigation was supported in part by funds provided to WSU through the NTH Biomedical Research Grant and Grant-in-aid from Washington State University.